Sample introduction system for a flow cytometer

ABSTRACT

A sample introduction system for a flow cytometer allows easy change of sample containers such as test tubes and facilitates use in high pressure environments. The sample container includes a cap having a pressure supply chamber and a sample container attachment cavity. A sample container may be automatically positioned into the attachment cavity so as to sealably engage the end of the sample container as its outer surface. This positioning may be accomplished through some sample introduction mechanism. To facilitate cleaning, HPLC tubing and fittings may be used in a manner which facilitates removing of the entire tubing from both the nozzle container and other sample container cap to permit its replacement to avoid contamination. The sample container support may include horizontal stops which loosely limit the movement of the sample container and thus avoid further stresses upon it.

The U.S. Government has a paid-up license in this invention and thefight in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of a contractnumber DE-FG06-93ER-61662 awarded by the Department of Energy.

BACKGROUND OF THE INVENTION

This invention relates to flow cytometers which are designed forrepetitive processing of substances. Specifically, the invention relatesto systems which repetitively introduce samples without contaminationfor high speed processing. It is particularly suited to sorting andanalysis applications in both the clinical and research fields.

Flow cytometers have been in clinical and research use for many years.Basically, the systems act to position small amounts of a substancewithin a sheath fluid. This sheath fluid may either form droplets or mayexist within a transparent channel for optical analysis. Throughhydrodynamic focusing and laminar flow, the substance is split intoindividual cells and the like and is surrounded by a sheath fluid. Sinceindividual cells and the like are often processed, the speed at whichsubstance material can be processed is somewhat limited.

In many applications the substance involves a liquid which is frequentlychanged. This may include processing a sample from one patient toanother. These changes further limit the speed at which processing mayoccur. Further samples tested can also involve biologically activematerials such as viruses and the like. This can intensify the need tothoroughly clean and decontaminate the system when changing samples.

In use, it is typical to receive sample substances as liquids containedwithin test tubes. These test tubes are attached or inserted into a flowcytometer system which pressurizes the test tube and thus forces thesubstance through tubing and into a nozzle container of the flowcytometer. For convenience, the test tubes are often attached byinserting a stopper into the test tube top. These stoppers often had twotubes positioned through them: a tube for supplying pressure to the testtube and a tube for allowing the substance to flow out of the test tube.As indicated in U.S. Pat. No. 5,182,617, a variety of designs have beenproposed to facilitate more rapid processing. These designs have,however, not totally met the needs of those involved in the actualprocessing of samples.

One of the problems faced has been the stresses placed upon the testtube or other container as it is pressurized for use. Because of therelationship between the speed of processing and the pressure applied tothe sample container, this problem is intensified as higher processingspeeds are pursued. While naturally other containers other than a testtube could be used, the time required to transfer substance to some moreappropriate container and the additional decontamination potentiallyrequired has made such solutions generally undesirable. In someapplications, those involved have simply utilized the conventionalcomponents closer to their inherent limitations and may have evenlimited the performance of the flow cytometer to accommodate suchlimitations. Obviously, such an approach, although practical, isundesirable.

Yet another problem faced by those skilled in the art is the fact thatthe proper combination of features and performance for practical use ofa flow cytometer for repetitive sampling of substances has not yet beenachieved. Apart from the actual analysis capability of the flowcytometer, it is necessary to provide a system which those involved inthe actual usage of the system find convenient. For some applications,this involves providing a system which can be easily cleaned ordecontaminated between sample runs. In others an easily manipulatableand inexpensive system may be desired. The present invention combinesfeatures to achieve a practicably implementable system which meets eachof these needs (and other needs more appropriately).

As mentioned, when biologically active materials are used, the problemof contamination is of greater concern. Not only has this mandated morethorough cleaning of the system between uses, but it also has made theproblem of aerosols more acute. As pressure is applied to the samplecontainer, this pressure--and the air flows associated with it--canresult in the creation of aerosols which may contain a biologicallyactive substance. Naturally, this must be avoided. Through potentiallyindependent features, the present invention acts to minimize thecreation of the aerosols in the first place.

As explained, a number of the foregoing problems have long beenrecognized by those having ordinary skill in the art. Solutions,however, have not been achieved in a practical and efficient manner eventhough the implementing arts have long been available. To some extent,this may be due to the fact that those having ordinary skill in the artmay not have fully appreciated the nature of the problems or may havesimply failed to consider designs which could be practically adapted totraditional components. Indeed, the directions taken by those skilled inthe art have to some degree been directed away from the directions takenin the present invention. Until the present invention a system whichsimultaneously met the needs for high speed processing for changingsamples, for cleaning the system, and for avoiding contamination was notpractically available for many applications.

SUMMARY OF THE INVENTION

The present invention involves improvements which may be implemented inconventional systems in a variety of ways. As to one feature, theinvention discloses a system which acts to seal the flow cytometeraround the exterior of the sample container to avoid and minimizes theeffects of the stresses placed upon such a container when it ispressurized. This system is provided through an operating mechanismwhich both facilitates use and may be easily disassembled and cleaned.The operating mechanism is designed to accommodate high pressurefittings in a manner which allows for the complete removal, disposal,and/or replacement of tubing components and the like to further minimizecontamination concerns. Each of these are designed to facilitate the useof higher pressures as may be associated with high speed processing. Inaddition, the sample container cap (which is designed to seal around theexterior of a test tube and the like) is designed so as to applypressure in a manner which minimizes the possibility of the creation ofaerosols from the substance used by being angled away from thesubstance.

Accordingly, it is an object of the invention to facilitate the use oftraditional components in more demanding applications. In keeping withthis object, a goal is to provide designs which reduce the stresses onsample containers such as test tubes and the like. It is also an objectto provide for a system which is capable of use at higher pressures andhigher processing speeds. The design is configured so as to minimizesthe limitations placed upon systems as a result of the structure of thecomponents utilized. The design also has as a goal accommodating theunavoidable movement of the sample container during the processingprocedure.

Another object of the invention is to reduce--and eliminate wherepossible--the possibility of aerosols being created as a result of thesample introduction facet of the processing procedure. Thus a goal is toallow the use of a pressure feed system which does not act to createaerosols when in use. A goal is also to direct the pressure away fromthe substance so that gas flows tend not to exist at the surface of thesubstance.

A broadly stated object of the invention is to provide a system which iseasy for the operator to use. In keeping with this object, a goal is toallow for one-step operation where possible when attaching the samplecontainer. Another goal is to allow for easy cleaning anddecontamination of the entire system. Thus a goal is to provide for asystem which may be easily disassembled and which includes replaceableand disposable components.

Naturally, further objects of the invention are disclosed throughoutother areas of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross-sectional view of a sample introductionsystem according to the present invention as applied to a droplet flowcytometer.

FIG. 2 is a diagram of a conventional test tube introduction design.

FIG. 3 is a side view of the embodiment of FIG. 1 showing the capraising mechanism operation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As can be seen from the drawings and in keeping with the objects of theinvention, the basic concepts of the present invention are easilyimplemented. FIG. 1 shows one design through which this is possible. Asit shows, the flow cytometer (1) incudes a sample introduction system(41) and a nozzle system (42). Generally, sample introduction system(41) acts to provide the appropriate flow of some sample material tonozzle system (42) so as to permit processing. As is well understood bythose having ordinary skill in the art, this processing typicallyinvolves the introduction of a sheath fluid through sheath fluid port(4) into nozzle volume (3) from some sheath reservoir (5). This sheathfluid has introduced to it the substance through substance introductionport (9). Together, the substance and the sheath fluid may then exitnozzle container (2) in jet (12) so as to form droplets (13) forprocessing. This processing may include sorting through the differentialapplication of charge to the various droplets (13) as those skilled inthe art readily understand. Additionally, it should be understood thatalthough FIG. 1 shows sample introduction system (41) as attached tonozzle system (42) configured as a droplet flow cytometer, naturally,sample introduction (41) can be applied to other flow cytometer designsas well. This would include channel type flow cytometers as thoseskilled in the art would readily understand. Such systems may act todirect flow within a sealed channel or may cause a continuous jet toflow without forming into regular droplets

As shown in FIG. 1, the particular configuration of sample introductionsystem (41) has a number of features. First, it can be seen that samplecontainer (8) includes the test tube configuration shown. Such acontainer may be made of plastic, glass, or any other material. Samplecontainer (8) is attached to substance supply tube (45) through use of asample container cap (43) which is situated over top end (44) of samplecontainer (8).

In order to introduce the substance, sample container cap (43) hasattached to it, substance supply tube (45). [It may also have a separateport for hypodermic insertion of a substance as shown in FIG. 1,however, this element is not an aspect of the present invention.] Asshown, substance supply tube (45) may be positioned on the vertical axisof sample container cap (43) so that second end (46) of substance supplytube (45) is positioned into sample container (8). In this fashion whenpressure is applied to sample container (8), the substance is forcedthrough second end (46) of substance supply tube (45) so as to pass intonozzle volume (3) and be emitted from substance introduction port (9).This substance introduction port (9) may actually be the end of astainless steel tube which is attached to first end (47) of substancesupply tube (45).

As shown, sample container cap (43) is configured to have samplecontainer (8) easily attached to it. To facilitate this, samplecontainer cap (43) has an inner surface which forms sample containerattachment cavity (48). This attachment cavity (48) may be positionedabout top end (44) of sample container (8) so as to sealably engage theouter surface of sample container (8). This sealing effectivelyestablishes substance supply tube (45) sealed to sample container (8).In the design shown, this may be accomplished through the use of O-ring(49) which acts as an external seal. In this fashion, when pressure isapplied to sample container (8) it does not urge sample container (8)away from the seal as in conventional designs but rather urges it in atighter fashion against the external seal. Thus by providing O-ring (49)positioned on the inner surface of sample container cap (43) and againstthe outer surface of sample container (8), the pressure applied bypressure source (50) actually can enhance the seal. Further, in sealingsubstance supply tube (45) to sample container (8), sample container cap(43) has substance supply tube (45) attached to it through removabletubing seal (51). Through this design, the entire length of substancesupply tube (45) may be removed, replaced, or cleaned.

With this understanding, it can be seen how the present design differsin one regard from the prior art. Referring to FIG. 2, it can be seenhow the prior art traditionally involves stopper (52) through whichsubstance supply tube (45) passes. Upon applying pressure throughpressure tube (53), sample container (8) is urged away from stopper(52). In sharp contrast, the present design accomplishes exactly theopposite, namely, upon applying pressure through angled pressure source(54) (the angled aspect of this element is discussed later) samplecontainer (8) is urged against O-ring seal (49) and thus the seal isactually enhanced by the pressure. Additionally, the stresses onconventional designs are caused by both the pressure and stopper (52) ina manner which compounds. Again, in sharp contrast, the stresses onsample container (8) of the present invention oppose each other and thusminimize the effect upon it.

Referring again to FIG. 1, it can be seen how even when in use thedesign is less delicate. This is in part through the design of samplecontainer support (55). As shown, sample container support (55) isdesigned to receive sample container (8) and support it vertically.Thus, sample container support (55) engages the bottom of samplecontainer (8) and supports it along the vertical axis by being situatedbelow sample container cap (43). Although not a requirement, it may bedesirable for some applications to design sample container support (55)so as to include horizontal stops (56) which are generally detached fromsample container (8) when it is inserted. Horizontal stops (56) can thusallow some limited movement in the horizontal plane and thus act toloosely limit sample container (8). This can be advantageous sincesample container (8) may be a delicate component such as a glass testtube. In this fashion, whether sample container cap (43) is operatedmanually, remotely, or through some mechanism, unnecessary stresses onsample container (8) can be avoided to an even greater degree.

As mentioned earlier, two of the goals of the invention includeproviding a design which can be easily cleaned and decontaminated aswell as a design which facilitates the use of higher pressures forfaster processing. Each of these are accommodated through the designsshown. First, through the selection of tubing and fittings such as thoseused in high pressure liquid chromatography (HPLC) applications, the useof high pressures is possible. Second, these designs (as well as anyothers selected) are accommodated in a manner so as to allow substancesupply tube (45) to be removable from the components. Thus, samplecontainer cap (43) is designed to allow attachment and detachment ofsubstance supply tube (45) at removable tubing seal (51). Similarly,nozzle container (2) is also designed to accommodate the same type ofremovable tube seal (51). When changing substances, it is now possibleto remove all of substance supply tube (45) and replace it. This greatlyfacilitates decontamination as it is very difficult to thoroughly cleansubstance supply tube (45) through back flushing and the like.Additionally, the design of substance supply cap (43) permits tubing tobe attached at removable tube seal (51) while simultaneously allowingsubstance supply tubing (45) to pass through sample container cap (43)and down into sample container (8). Through these features not only cansubstance supply tubing (45) be replaced but also sample container cap(43) may be removed from the flow cytometer for decontamination as wellas nozzle container (2) and substance introduction port (9). Each ofthese components may then be chemically and/or heat treated for easierdecontamination.

Referring to the conventional design shown in FIG. 2, it can beunderstood how the introduction of pressure through pressure tube (53)can cause the creation of aerosols within sample container (8). Whilethis problem seems to have a very simple solution, in fact, thoseskilled in the art had not, prior to the present invention, practicallyachieved such solutions. Instead, the use of stopper (52) lent itself tothe inclusion of pressure tube (53) in such a manner so as to allow gasflows from this pressure to impinge directly upon the substancecontained within sample container (8). To the contrary, the design shownin FIG. 1 avoids this aspect. As shown, sample container cap (43) hasincluded on its inner surface angled pressure source (54) which directsair into pressure supply chamber (57). In a basic form, the pressure isapplied at an angle to the vertical axis of sample introduction system(41) (and likewise to sample container cap (43)) so as to avoid anydirect gas flow onto the substance contained within sample container(8). As shown, the angle at which the gas flows supplying the pressureare emitted may be about 90 degrees to that of the vertical axis ofsample container cap (43). Further, to additionally minimize the gasflows experienced at the surface of the substance within samplecontainer (8), pressure supply chamber (57) may be baffled or otherwisedesigned. As shown, it is simply enlarged so as to avoid having a narrowstream of gas move toward the substance.

Finally, referring to FIG. 3, sample introduction mechanism (58) may beeasily understood. As shown sample introduction mechanism (58) mayinclude one or more arms (59) which attach to the outer surface ofsample container support (55) at one or more attachment points (60). Byoperating sample introduction mechanism (58) sample container cap (43)may be engaged by the top of sample container (8) to allow easy samplechanging. Naturally this mechanism may be designed in a host ofdifferent fashions, may be automated, or may be manually operated. Suchdesigns would include configurations which would tend to automaticallyattach and hold sample container during the removal process as well asdesigns which might purposely move sample container support (55)parallel to its vertical axis initially so as to avoid any additionalstresses upon sample container (8).

The foregoing discussion and the claims which follow describe thepreferred embodiment of the present invention. Particularly with respectto the claims it should be understood that changes may be made withoutdeparting from the essence of this patented invention. It is intendedthat such changes are permissible to accommodate varying applicationsand will still fall within the scope of this patent as it is simply notpractical to describe and claim all possible revisions to the design. Tothe extent such revisions utilize the essence of the invention eachwould naturally fall within the breadth of protection encompassed bythis patent.

I claim:
 1. A method of introducing a sample for flow cytometerprocessing comprising the steps of:a. establishing a nozzle volume; b.attaching a substance supply tube to said nozzle volume; c. attaching asample container having an outer surface to said substance supply tube,said sample container serving as a reservoir for a substance to besupplied via the supply tube to the nozzle volume; d. sealing saidsample container to said substance supply tube by a seal which engagessaid sample container about said outer surface; e. introducing a flow ofsheath fluid into said nozzle volume; f. applying a pressure to saidsample container; and g. allowing said substance to flow from saidsample container to said sheath fluid in said nozzle volume.
 2. A methodof introducing a sample for flow cytometer processing as described inclaim 1 wherein said sample container is attached to a sample containercap and wherein said step of attaching a sample container comprises thestep of operating a sample introduction mechanism.
 3. A method ofintroducing a sample for flow cytometer processing as described in claim2 wherein said step of attaching said sample container further comprisesthe steps of:a. supporting said sample container vertically; while b.loosely limiting said sample container so as to permit some movement ofsaid sample container horizontally.
 4. A method of introducing a samplefor flow cytometer processing as described in claim 1 wherein saidsample container has an axis and wherein said step of applying apressure to said sample container comprises the step of applying saidpressure through an element oriented at an angle to said axis.
 5. Amethod of introducing a sample for flow cytometer processing asdescribed in claim 4 wherein the angle is about 90 degrees.
 6. A methodof introducing a sample for flow cytometer processing as described inclaim 4 wherein said axis is vertical.
 7. A method of introducing asample for flow cytometer processing comprising the steps of:a.establishing a nozzle volume; b. attaching a substance supply tube tosaid nozzle volume; c. attaching a sample container having an axis tosaid substance supply tube, said sample container serving as a reservoirfor a substance to be supplied via the supply tube to the nozzle volume;d. introducing a flow of sheath fluid into said nozzle volume; e.applying a pressure to said sample container through an element orientedat an angle to said axis; and f. allowing said substance to flow fromsaid sample container to said sheath fluid in said nozzle volume.
 8. Amethod of introducing a sample for flow cytometer processing asdescribed in claim 7 wherein the angle is about 90 degrees.
 9. A methodof introducing a sample for flow cytometer processing as described inclaim 7 wherein said axis is vertical.